Ion transporters are more difficult to study than ion channels, and their molecular function is intrinsically more complex. Therefore, we developed sensitive new ion selective electrode (ISE) methods to quantify ion fluxes per se during patch clamp recording. We will exploit these methods to study the three most active Na transporters in cardiac myocytes-Na/Ca exchange (NCX1), Na/K pumps, and Na/H exchange (NHE1). Together, these transporters are important determinants of normal cardiac function, as well as myocyte fate during ischemia. (1) Na/Ca exchange (NCX1). Preliminary data show that NCX1 function is modified by a Ca-dependent Na transport slippage, probably occurring as cotransport of lNa+ 1Ca. We will now probe whether the slippage function is regulated by protein kinases, signaling lipids, and cell volume changes. And we will explore the role of NCX1 slippage in determining cytoplasmic Na and pacemaking currents. (2) Na/K pump. Preliminary data show that Na/K pumps do not operate by a perfect 3Na/2K stoichiometry. We will test multiple explanations for the non-integer coupling, measuring Na flux/current ratios, Kflux/current ratios, and Na/K pump reversal potentials under appropriate conditions. We will test whether flux coupling is affected by pump phosphorylation by PKA and PKC, and we will compare ion flux coupling of alpha1 and alpha2 isoforms. (3) Na/H exchange (NHE1). Preliminary data show that NHE1 activity remains highly robust and volume-sensitive in whole-cell recording with extensive dialysis of non-hydrolyzable ATP (AMP-PNP) and with disruption of actin cytoskeleton. We will characterize cis/trans interactions of Na and H concentrations in determining NHE1 activity, the dependence of NHE1 activity on nucleotides and phosphoinositides, and we will test our working hypothesis that cell volume changes act on NHE1 by a direct mechanical mechanism. We will probe the basis for NHE1 mechano-sensitivity and the reasons for complete loss of NHE1 activity in excised patches. (4) In parallel with the experimental program, we will develop improved mathematical models of cardiac ion homeostasis with an emphasis on these three Na transporters.